Nanopore device with graphene supported artificial lipid membrane

ABSTRACT

The invention features the use of graphene, a one atom thick planar sheet of bonded carbon atoms, in the formation of artificial lipid membranes. The invention also features the use of these membranes to detect the properties of polymers (e.g., the sequence of a nucleic acid) and identify transmembrane protein-interacting compounds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the National Stage of International Application No.PCT/US2011/039621, filed on Jun. 8, 2011, which claims benefit to U.S.Provisional Application Nos. 61/352,636 and 61/352,791, filed Jun. 8,2010, each of which is hereby incorporated by reference.

STATEMENT AS TO FEDERALLY FUNDED RESEARCH

This invention was made with government support under HG003703 awardedby National Institutes of Health. The government has certain rights inthe invention.

BACKGROUND OF THE INVENTION

The invention relates to the field of artificial lipid bilayermembranes.

Artificial monolayers and bilayers of lipids are frequently used in thediscovery of the physiological and pharmacological properties of cellsurface and intracellular membrane proteins. Artificial monolayers andbilayers are also key components of many commercial devices includingsensitive biosensors that are being used or developed to detectbiological warfare agents, to discover membrane receptors that regulatehuman disease or pathogen function, and to screen pharmaceutical agentsto reveal their function, identity, and concentration. During the pastforty years, electrophysiological studies of proteins reconstituted intounsupported lipid bilayers have generated detailed information onmembrane protein function, ligand-binding, and kinetics. Because thefunction of membrane proteins plays a critical role in all aspects ofdevelopment, organ function, and health, studies of proteinsreconstituted into lipid bilayers are extensively used to screen forpotentially useful drugs as well as to identify targets for drugtherapies.

Electrophysiological studies have been used to characterize theproperties of single molecules, such as DNA molecules, that can betranslocated through transmembrane proteins such as α-hemolysin.

While monolayers are often formed and supported on hydrophobic surfaces,bilayers are more frequently assembled as free-standing membranes. Lipidmonolayers and bilayers are labile, delicate structures that are easilydamaged. Free-standing bilayer membranes, which are particularly wellsuited for acquisition of electrophysiological data from membraneproteins and sensitive nanopore detection methods, are especiallysusceptible to rupture. Therefore, there exists a need in the art forresilient artificial lipid membranes.

SUMMARY OF THE INVENTION

In general, the invention features a device including an artificialmembrane on graphene with one or more apertures, e.g., having adimension of less than 10 μm (e.g., a plurality of apertures arranged inan array). The artificial membrane includes two lipid monolayers, eachcontacting one face of the graphene, and the two lipid monolayers form alipid bilayer within an aperture in the graphene. The lipid bilayer canfurther include a protein (e.g., a transmembrane protein). Thetransmembrane protein can be an ion channel, a multidrug resistancepump, a cytokine receptor, a receptor from the immunoglobin superfamily,a receptor from the tumor necrosis factor receptor family, a chemokinereceptor, a receptor tyrosine kinases, or a TGF beta receptor.

The device can also include at least one pore (e.g., α-haemolysin)disposed in at least one aperture, with, e.g., at most one pore disposedin each of the apertures. A molecule motor can be adjacent to at leastone of the pores and can be capable of moving a polymer with respect tothe pore. In embodiments where the polymer is a nucleic acid, examplesof molecular motors are a DNA polymerase (e.g., E. coli DNA polymeraseI, E. coli DNA polymerase I Large Fragment (Klenow fragment), phage T7DNA polymerase, Phi-29 DNA polymerase, Thermus aquaticus (Taq) DNApolymerase, Thermus flavus (Tfl) DNA polymerase, Thermus Thermophilus(Tth) DNA polymerase, Thermococcus litoralis (Tli) DNA polymerase,Pyrococcus furiosus (Pfu) DNA polymerase, Vent™ DNA polymerase, Bacillusstearothermophilus (Bst) DNA polymerase, AMV reverse transcriptase, MMLVreverse transcriptase, and HIV-1 reverse transcriptase), an RNApolymerase (e.g., T7 RNA polymerase, T3 RNA polymerase, SP6 RNApolymerase, and E. coli RNA polymerase), a ribosome, an exonuclease(e.g., exonuclease Lambda, T7 Exonuclease, Exo III, RecJ₁ Exonuclease,Exo I, and Exo T), or a helicase (e.g., E-coli bacteriophage T7 gp4 andT4 gp41 gene proteins, E. coli protein DnaB, E. coli protein RuvB, andE. coli protein rho).

In any of the foregoing, the device can further including a frame (e.g.,a non-conductive frame including a ceramic or silicon nitride)supporting the graphene and/or first and second electrodes disposed onopposite sides of the graphene. The frame can be positioned on one faceof the graphene forming a compartment for housing liquid for each of theapertures. Multiple compartments can each include an electrode (or othersensor) and are typically fluidically isolated.

In another aspect, the invention features a method of analyzing a targetpolymer including introducing the target polymer to a pore-containingdevice of the invention, allowing the target polymer to move withrespect to the pore to produce a signal, and monitoring the signalcorresponding to the movement of the target polymer. Exemplary devicesused in these methods include a molecular motor, e.g., an exonuclease,to control movement of the polymer, e.g., a nucleic acid. The signalmonitoring can include measuring a monomer-dependent characteristic(e.g., identity of a monomer or the number of monomers in the polymer)of the target polymer. In one embodiment, the rate of movement of thepolymer can be altered before, during, or after the signal monitoring.The method may alternatively be used to study the effect of the polymeron the pore or molecular motor.

In another aspect, the invention features a method of identifying atransmembrane protein-interacting compound by contacting a transmembraneprotein positioned in any of the foregoing devices with a compound andmeasuring at least one activity of the protein (directly or indirectlyby its effect on the compound) in the presence of the compound, wherebya change in activity in the presence of the compound indicates that thecompound interacts with the protein. Compounds may bind to, inhibit,agonize, react with, or be altered during an interaction with theprotein. Alternatively, the compound may affect the integrity orrigidity of the bilayer, thereby indirectly interacting with theprotein. In one embodiment, an electric field is applied and themeasuring at least one activity of the transmembrane protein includesmeasuring the electrical resistance of the transmembrane protein.

Similarly, the methods can be used to measure the interaction ofcompounds with other membrane molecules, e.g., a lipid in the bilayer ora compound conjugated to a lipid. In such methods, the lipid layer mayor may not include a pore.

By “across” is meant a measurement or movement not from one face to theother, e.g., in substantially the same plane as a face of graphene.

By “through” is meant a measurement or movement from one face to theopposing face of graphene.

By a “change in activity” is meant a measured difference in a propertyof an aperture in the presence of a candidate compound when compared tothat same property of the aperture in the absence of the candidatecompound. A change in activity can be, e.g., an increase or decrease of1%, 5%, 10%, 25%, 50%, 75%, 100%, or more.

By “cis” is meant the side of an aperture through which a polymer entersthe aperture or across the face of which the polymer moves.

By “trans” is meant the side of an aperture through which a polymer (orfragments thereof) exits the aperture or across the face of which thepolymer does not move.

By “molecular motor” is meant a molecule (e.g., an enzyme) thatphysically interacts with a polymer, e.g., a polynucleotide, and iscapable of physically moving the polymer with respect to a fixedlocation. Although not intending to be bound by theory, molecular motorsutilize chemical energy to generate mechanical force. The molecularmotor may interact with each monomer of a polymer in a sequentialmanner.

Other features and advantages of the invention will be apparent from thefollowing drawings, detailed description, and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic showing the creation of a lipid bilayer byadapting the method of Montal and Muller (Proc Nat Acad Sci 69,3561-3566 (1972)) for use on graphene.

FIG. 2 is a schematic diagram of graphene mounted on a frame such thatboth faces of the graphene are accessible. Inset: similarly mountedgraphene into which a single aperture has been drilled.

FIG. 3 is a schematic diagram of a structure for forming an array ofgraphene-supported lipid bilayers (not to scale). A 2×3 array of sixnanopores is shown, each of which supports a lipid bilayer. Eachcompartment in the underlying non-conductive ceramic frame may form aseparate solution compartment equipped with an electrode (e.g.,Ag—AgCl), with a common ground electrode on the opposite side of thegraphene supported bilayers.

FIG. 4 is a schematic showing α-hemolysin, a trans-membrane protein,inserted into a lipid bilayer through an aperture in graphene. If theaperture is drilled to an appropriate dimension (for α-hemolysin ca. 5nm), its proximity to the stem of the transmembrane protein (arrows)makes it possible for a highly localized field to be applied around thetransmembrane portion of the inserted protein and makes it furtherpossible for conformational and other functional protein alterations tobe sensed by changes in the graphene's in-plane conductivity and/orother electrical properties.

FIG. 5 is a schematic diagram of the use of membrane fusion to form agraphene-supported bilayer.

FIG. 6 is a graph showing current as a function of voltage across an 8nm aperture in graphene under the indicated conditions.

FIG. 7 is a schematic diagram of graphene mounted on a SiN_(x)/Si frame.Portions of the graphene near the aperture have been ablated. Thediagram also shows two electrodes mounted on the same face of thegraphene.

DETAILED DESCRIPTION OF THE INVENTION

Graphene is a one atom thick planar sheet of bonded carbon atoms. Wehave discovered that graphene is a superior hydrophobic support for thegeneration of artificial lipid monolayers and bilayers because, e.g.,graphene can be fabricated into sheets that are atomically thin, strong,relatively chemically inert, and mechanically stable. Although graphenedoes not conduct ions or electrons from one face to the other, it iselectrically conductive in the plane of each face. This in-planeconductivity is sensitive to the environment to which the graphene isexposed.

Graphene can be fashioned to contain small apertures, e.g., having adimension of millimeter to single nanometer (or less) across, that allowthe formation of lipid bilayers. Each face of graphene supports a lipidmonolayer, and two monolayers may contact and form lipid bilayers inapertures in the graphene. Because graphene is atomically thin, thedislocation between the graphene supported lipid molecules on each ofits faces and the lipid molecules forming a lipid bilayer is minimal.This configuration creates a robust, giga-ohm seal between the bilayerand the graphene.

Molecules (e.g., membrane proteins) can be incorporated into a bilayerin an aperture in graphene. Such structures are useful for investigatingand determining the structural and functional properties of, e.g.,incorporated membrane proteins. Also, pores can be incorporated into abilayer aperture in graphene. Such structures are useful forinvestigating and determining the structure of polymers (e.g., thesequence of a nucleic acid). In order to monitor such structural andfunctional properties, the invention may include suitably patterned,electrically connected graphene that can transmit electrical currents,including in plane current measurements such as tunneling currents, totraverse the central hydrophobic region of the transmembrane proteins tomonitor or alter membrane activity.

Alternatively, a lipid monolayer may be formed on one face of grapheneby having oil or another hydrophobic medium on one side of the apertureand water or another hydrophilic medium on the other side. A lipidmonolayer within an aperture in graphene would then separate thehydrophobic and hydrophilic media. The interaction of molecules withthis interface between media can be measured, e.g., using electrical oroptical detection as described herein.

Lipid Bilayers

The invention features lipid bilayers disposed within grapheneapertures. In one method of making bilayers, lipid monolayers aredisposed on both faces of graphene resulting in a lipid bilayerspontaneously forming within apertures in the graphene. In anothermethod, liposomes disposed on both sides of the graphene are induced torearrange into lipid bilayers within apertures.

In one embodiment, a bilayer can be created using a procedure adaptedfrom Montal and Muller (Proc Nat Acad Sci U.S.A. 69, 3561-3566 (1972)).In the present invention graphene, which can be supported on a rigidframe, is slowly immersed into a solution in a Langmuir-Blodgett troughwith a solution-air interface covered with a lipid monolayer maintainedat constant pressure (see FIG. 1). FIGS. 2 and 3 show graphene mountedon a suitable frame. As the graphene mounted on its frame penetratesthrough the lipid-covered solution-air interface, a monolayer of lipidis transferred to each of the two faces of the graphene. Furthermore, alipid bilayer (whose leaflets are continuous with the two leaflets nowcovering each side of the graphene) can be induced to self assembleacross apertures (nano to micro in size) in the graphene. The graphenecan be supported by a frame to form two aqueous compartments. Here, alipid bilayer in the graphene aperture will represent the thinnestportion separating the two aqueous compartments.

In another embodiment, a graphene supported bilayer is created byexposing both faces of the graphene to solutions containing unilamellarliposomes whose diameters are greater than the diameter of an aperturein the graphene. Because intact liposomes (whose outer surfaces arehydrophilic) are unlikely to bind to the hydrophobic graphene surface,some liposomes fuse with each other across the apertures in the graphene(FIG. 5, top). This fusion results in molecular rearrangements andstructural disruptions creating a monolayer of lipid on each face of thegraphene and a bilayer within an aperture (FIG. 5, bottom).

In this embodiment, triggered fusion may be desirable so that the intactpre-fusion liposomes can be introduced on both sides of the graphenemembrane in a solution prior to the addition of the triggering agent(e.g., Ca⁺⁺). Millimolar concentrations of Ca⁺⁺ can be added toliposomes composed of 1:1 mole ratios ofphospatidylethanolamine:phosphatidylserine to trigger the fusion of twoliposomes. Bilayers and monolayers of lipid could also be formed on onlyone face of the graphene by standard methods known in the art, e.g., asdisclosed in Cooper (J. Mol. Recognit. 17, 286-315 (2004)).

The lipid bilayers can include saturated and unsaturated lipids.Additional examples of lipids are phospholipids (e.g.,phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, andphosphatidylglycerol), glycolipids, and sphingolipids (e.g.,sphingomylein and sterols). Lipids may also be conjugated to othermolecules, e.g., at the polar head.

The bilayers are artificial in the sense that they are not attached to aliving cell. Membranes or components from an organism, e.g., eukaryotic(e.g., plant, fungal, protist, or animal), prokaryotic, or virus may ormay not be employed.

Graphene Apertures

Apertures in graphene are, e.g., nanometer- to micron-sized holes.Apertures useful in the invention typically range in width, i.e., acrossthe aperture, from 1-1000 nm, e.g., at most 750, 500, 400, 300, 250,200, 150, 100, 90, 80, 70, 60 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3,or 2 nm and at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70,80, 90, 100, 150, 200, 250, 300, 400, 500, or 750 nm. The throughdimension is controlled by the thickness of the graphene. This thicknesscan be increased by employing a stack of graphene sheets, e.g., 2-5sheets. The dimensions and shapes of the aperture employed will dependon the intended application.

In certain embodiments, an aperture is sized to maximize the proximityand contacts between a transmembrane protein and the graphene. Forexample, an aperture between 4.5-5.5 nm across is suitable toaccommodate the 4.4 nm diameter transmembrane stem of α-hemolysin (FIG.4). Thus, an aperture may have a dimension that is no more than 5, 4, 3,2, 1, 0.5, 0.25, or 0.1 nm wider than the diameter of a transmembraneprotein. Excess graphene in the region of the aperture may also beremoved with a focused electron beam or ion beam, as shown in FIG. 7.Thus, in certain embodiments, the aperture is disposed in a region ofthe graphene that is no more than 0.5, 1, 2, 3, 4, 5, 10, 20, 30, 40, or50 nm wider than the aperture.

Apertures can be sized to accommodate the passage of particularpolymers, e.g., polynucleotides, polypeptides, and polysaccharides. Forexample, an aperture of approximately 0.5-2.0 nm across is suitable forthe passage of single stranded DNA; an aperture of 1.0-3.0 nm across issuitable for the passage of double stranded DNA; and an aperture of1.0-4.0 nm across is suitable for the passage of polypeptides.

Because transmembrane proteins can fully insert or self-assemble only inbilayer regions, an array of apertures in the graphene will create anarray of lipid bilayers containing membrane proteins inserted into thesebilayers in the same pattern with the same intermolecular spacing as thearray of graphene apertures. Furthermore, because graphene can beelectrically contacted and is electrically conductive within the planeof each face, the central hydrophobic region of the transmembraneproteins inserted into these unsupported lipid bilayers can be subjectedto highly localized electrical fields without disrupting the lipidenvironment (FIG. 4).

Because of the strength and mechanical stability of graphene, aperturesa single nanometer across can be fabricated (using, e.g., electron orion beams) either singly or in arrays (FIG. 3), with any desiredspacing, and are capable of supporting stable lipid bilayers.

Membrane Molecules

Molecules may be disposed within the lipid bilayers formed in aperturesin graphene. The molecules can be pore-forming proteins (e.g., ionchannels and voltage gated channels) or transmembrane proteins (e.g.,transmembrane receptors).

Examples of pore-forming proteins that can be used in the inventioninclude Gramicidin (e.g., Gramicidin A from Bacillus brevis; availablefrom Fluka, Ronkonkoma, N.Y.); LamB (maltoporin), OmpF, OmpC, or PhoEfrom E. coli, Shigella, and other Enterobacteriaceae, α-hemolysin (fromS. aureus), Tsx, the F-pilus, lambda exonuclease, MspA, andmitochondrial porin (VDAC). Homologs and paralogs of these pores mayalso be employed.

A modified voltage-gated channel can also be used in the invention.Voltage gated channels include voltage-dependent calcium channels,voltage-gated sodium channels, voltage-gated potassium channels, andvoltage-gated proton channels. Methods to alter inactivationcharacteristics of voltage gated channels are well known in the art (seee.g., Patton, et al., Proc. Natl. Acad. Sci. USA, 89:10905-09 (1992);West, et al., Proc. Natl. Acad. Sci. USA, 89:10910-14 (1992); Auld, etal., Proc. Natl. Acad. Sci. USA, 87:323-27 (1990); Lopez, et al.,Neuron, 7:327-36 (1991); Hoshi, et al., Neuron, 7:547-56 (1991); Hoshi,et al., Science, 250:533-38 (1990), all hereby incorporated byreference).

Pores can be formed in a lipid bilayer using chemicals (or peptides)such as Nystatin, as is well known in the art of whole-cell patchclamping (“perforated patch” technique), and peptide channels such asAlamethicin.

Pore-forming proteins can also be linked to molecular motors (e.g., apolymerase or exonuclease). Here, synthetic/recombinant DNA coding for afusion protein can be transcribed and translated, then inserted into thelipid bilayer region of the artificial membranes of the invention. Forexample, the C-terminus of E. coli DNA polymerase I (and by homology, T7DNA polymerase) is very close to the surface of the major groove of thenewly synthesized DNA. If the C-terminus of a polymerase is fused to theN-terminus of a pore forming protein such as colicin E1 and the colicinis inserted into an artificial membrane, one opening of the colicin poreshould face the DNA's major groove and one should face the opposite sideof the lipid bilayer. The colicin molecule can be modified to achieve apH optimum compatible with the polymerase as in Shiver et al. (J. Biol.Chem., 262:14273-14281 1987, hereby incorporated by reference). Bothpore and polymerase domains can be modified to contain cysteinereplacements at points such that disulfide bridges form to stabilize ageometry that forces the pore opening closer to the major groove surfaceand steadies the polymer as it passes the pore opening. The loops of thepore domain at this surface can be systematically modified to maximizesensitivity to changes in the DNA sequence.

Transmembrane receptors include cytokine receptors (e.g., IL-2 receptor,interferon alpha/beta receptor, interferon gamma receptor,erythropoietin receptor, GM-CSF receptor, G-CSF receptor, growth hormonereceptor, prolactin receptor, oncostatin M receptor, and leukemiainhibitory factor receptor), members of the immunoglobin superfamily(e.g., interleukin-1 receptor, CSF1, C-kit receptor, and interleukin 18receptor), members of the tumor necrosis factor receptor family (e.g.,CD27, CD30, CD40, CD120, and lymphotoxin beta receptor), chemokinereceptors (interleukin-8 receptor, CCR1, CXR4, MCAF receptor, and NAP-2receptor), receptor tyrosine kinases (e.g., erythropoietin receptor,insulin receptor, Eph receptors, and insulin-like growth factor 1receptor), TGF beta receptors (e.g., TGF beta receptors 1 and 2), andother G-protein coupled receptors.

Additional molecules include ion channels and pumps, e.g., multidrugresistance pumps.

Molecular Motors

Any molecular motor that is capable of moving a polymer (e.g., apolynucleotide) of interest may be employed in the invention. Desirableproperties of a molecular motor include: sequential action, e.g.,addition or removal of one nucleotide per turnover; no backtrackingalong the target polymer; no slippage of the motor on the target polymerdue to forces, e.g., from an electric field, employed to drive a polymerto the motor; retention of catalytic function when disposed adjacent anaperture; high processivity, e.g., the ability to remain bound to atarget polymer and perform at least 1,000 rounds of catalysis beforedissociating.

Molecular motors include, e.g., polymerases (i.e., DNA and RNA),helicases, ribosomes, and exonucleases. The molecular motor that is usedaccording to the methods described herein will depend, in part, on thetype of target polymer being analyzed. For example, a molecular motorsuch as a DNA polymerase or a helicase is useful when the targetpolynucleotide is DNA, and a molecular motor such as RNA polymerase isuseful when the target polynucleotide is RNA. In addition, the molecularmotor used will depend, in part, on whether the target polynucleotide issingle-stranded or double-stranded. Those of ordinary skill in the artwould be able to identify the appropriate molecular motors usefulaccording to the particular application.

DNA polymerases have been demonstrated to function as efficientmolecular motors. Exemplary DNA polymerases include E. coli DNApolymerase I, E. coli DNA polymerase I Large Fragment (Klenow fragment),phage T7 DNA polymerase, Phi-29 DNA polymerase, thermophilic polymerases(e.g., Thermus aquaticus (Taq) DNA polymerase, Thermus flavus (Tfl) DNApolymerase, Thermus Thermophilus (Tth) DNA polymerase, Thermococcuslitoralis (Tli) DNA polymerase, Pyrococcus furiosus (Pfu) DNApolymerase, Vent™ DNA polymerase, or Bacillus stearothermophilus (Bst)DNA polymerase), and a reverse transcriptase (e.g., AMV reversetranscriptase, MMLV reverse transcriptase, or HIV-1 reversetranscriptase). Other suitable DNA polymerases are known in the art. Inone embodiment, approximately 300 nucleotides per second are threadedthrough the clamp of a DNA polymerase in a ratchet-like linear fashion,which decreases the probability of backward movement of thepolynucleotide. In certain embodiments, E. coli DNA polymerase I, theKlenow fragment, phage T7 DNA polymerase, Taq polymerase, and theStoffel fragment are excluded from the molecular motors employed in theinvention.

RNA polymerases, like DNA polymerases, can also function as efficientmolecular motors. Exemplary RNA polymerases include T7 RNA polymerase,T3 RNA polymerase, SP6 RNA polymerase, and E. coli RNA polymerases. Incertain embodiments, T7 RNA polymerase is excluded from the molecularmotors employed in the invention.

The molecular motor may also include a single-strand specific ordouble-strand specific exonuclease. Lambda exonuclease, which is itselfshaped as a pore with a dimension similar to α-hemolysin, can operate asa motor, controlling the movement of the nucleic acid polymer throughthe channel. Exonuclease Lambda is a trimeric enzyme isolated from theE. coli bacteriophage Lambda, and is particularly well suited for use inthe methods and devices of the invention for a number of reasons. Itacts upon double-stranded DNA, which is a preferred substrate forgenetic analysis. Also, it is a highly processive enzyme and acts upononly one strand of the double-stranded DNA, which facilitates themovement of a given DNA molecule with respect to an aperture. Further,the digestion rate is about 10-50 nucleotides per second (van Oijen etal. Science 301:1235 (2003); Perkins et al. Science 301:1914 (2003)). Asthe double stranded polymer passes through the pore, the exonucleasegrabs onto the 5′ single-stranded overhang of a first strand (viaendonuclease digestion or breathing of the double stranded DNA ends) andsequentially cleaves the complementary second strand at its 3′ end.During the sequential cleavage, the exonuclease progresses 5′ to 3′ downthe first strand, pulling the double stranded DNA through the channel ata controlled rate. Thus, the exonuclease can operate as a pore as wellas a motor for drawing the nucleic acid polymer through the channel.Exonuclease Lambda may also be excluded from the molecular motorsemployed in the invention. Additional exonucleases include, for example,T7 Exonuclease, Exo III, RecJ₁ Exonuclease, Exo I, and Exo T.

Another type of molecular motor is a helicase. Helicases are proteins,which move along polynucleotide backbones and unwind the polynucleotideso that the processes of DNA replication, repair, recombination,transcription, mRNA splicing, translation, and ribosomal assembly, cantake place. Helicases include both RNA and DNA helicases. Helicases havepreviously been described in U.S. Pat. No. 5,888,792. Exemplaryhelicases include hexameric helicases such as the E-coli bacteriophageT7 gp4 and T4 gp41 gene proteins, and the E. coli proteins DnaB, RuvB,and rho (for review see: West S C, Cell, 86, 177-180 (1996)). Hexamerichelicases unwind double stranded DNA in a 5′-3′ direction, which ensuresa directional analysis of the DNA target molecules. In addition, some ofthe helicases have processive translocation rates in excess of 1000nucleotides per second (Roman et al. J. Biol. Chem. 267:4207 (1992). Inaddition, these hexameric helicases form a ring structure having aninside hole dimension ranging in size from 2-4 nanometers and an outsidering dimension of about 14 nanometers, which is within the dimensionlimits of a useful molecular motor. The hexameric ring structure isformed and stabilized in the presence of Mg⁺² and some type ofnucleotide (NMP, NDP or NTP).

The intrinsic rate of movement of a particular molecular motor may bemodified, e.g., by chemical modification of the motor, by changes intemperature, pH, ionic strength, the presence of necessary cofactors,substrates, inhibitors, agonists, or antagonists, by the nature of themedium (e.g., the presence of nonaqueous solvents or the viscosity), byexternal fields (e.g., electric or magnetic fields), and hydrodynamicpressure. Such modifications may be used to start, stop, increase,decrease, or stabilize the rate of movement, which may occur during aparticular characterization. In addition, such modifications may be usedas switches, brakes, or accelerators, e.g., to start, stop, increase, ordecrease movement of a polynucleotide. In alternative embodiments,external forces (e.g., electric or magnetic fields or hydrodynamicpressure) generated other than by molecular motors may be used tocontrol the rate of movement. The rate of movement may be substantiallyslowed or even stopped (e.g., paused) before, during, or after analysisof a particular polynucleotide.

A molecular motor may be disposed on the cis or trans side of a graphenesupported lipid bilayer. In either case, the molecular motor can beadjacent the pore or inline with the pore. A molecular motor may also bewholly or partially disposed within a graphene aperture. A moleculemotor may be conjugated to a lipid in the bilayer or to another moleculedisposed in the bilayer, e.g., a non-pore forming protein or sterol.

Detection System

The invention features assays and devices for detecting properties ofcompounds using lipid bilayers. In one series of embodiments, thebilayers of the invention contain membrane molecules (e.g.,transmembrane proteins and pores). Compounds are then administered tothe bilayers and at least one property of the molecule is measured(directly or indirectly by the effect on the compound). Such propertiescan include electrical properties, e.g., effect on conductance. Inanother series of embodiments, the bilayers of the invention are used toidentify the properties of a polymer (e.g., the sequence or length of apolynucleotide). In this series of embodiments, a pore-forming moleculeis disposed in the lipid bilayer, and a property, e.g., electricalconductance across or through the pore, is measured as a polymer moveswith respect to the pore (e.g., by passing across or through the pore).The invention may also be employed in the study of organisms (e.g.,cells or viruses) with the bilayer or components thereof.

A typical detection system includes electronic equipment capable ofmeasuring characteristics of the compound, e.g., a polynucleotide, as itinteracts with the pore or other membrane molecules, a computer systemcapable of controlling the measurement of the characteristics andstoring or displaying the corresponding data, and one or more detectorscapable of measuring at least one property of compounds, pores, othermembrane molecules, or graphene in the device.

The detection system can measure transport properties, such as, but notlimited to, the amplitude or duration of individual conductance orelectron in plane current, such as tunneling current, changes across orthrough an aperture or pore.

In one embodiment, the detection system can be used to determine thenature or length of a polymer (e.g., a polynucleotide). Changes in theelectrical properties of a pore can identify the monomers in sequence,as each monomer has a characteristic conductance change signature, or achange in the conformation of a transmembrane protein. For instance, thevolume, shape, or charges on each monomer can affect conductance in acharacteristic way. Likewise, the size of the entire polymer can bedetermined by observing the length of time (duration) thatmonomer-dependent conductance changes occur. Alternatively, the numberof monomers in a polymer (also a measure of size) can be determined as afunction of the number of monomer-dependent conductance changes for agiven polymer traversing the aperture. The number of monomers may notcorrespond exactly to the number of conductance changes, because theremay be more than one conductance level change as each monomer of polymerpasses sequentially through or across the aperture. However, there is aproportional relationship between the two values that can be determinedby preparing a standard with a polymer having a known sequence. Otherdetection schemes are described herein.

Exemplary detection components have been described in WO 2009/045473, WO2009/045472, WO 2008/124107, WO 2007/084163, WO 06/052882, WO 06/028508,WO 05/061373, WO 04/077503, WO 03/003446, WO 03/000920, WO 01/42782, WO00/28312, and WO 00/79257, each of which is incorporated by reference inits entirety, and can include, but are not limited to, electrodesdirectly associated with the structure at or near the aperture, andelectrodes placed within the cis and trans pools. The electrodes can becapable of, e.g., detecting ionic current differences between the twopools or in plane currents, such as electron tunneling currents, acrossor through the pore or aperture. As described above, a frame can bedisposed to create discrete chambers on one or both faces of thegraphene, and each chamber may include an electrode. In this embodiment,the electrical property, e.g., conductance, of individual aperturesdisposed in an array can be separately measured allowing thesimultaneous detection of the activities associated with numerouspolymers or transmembrane proteins.

Time-dependent properties of the aperture may be measured by anysuitable technique. The properties may be a function of the medium usedin the assay, solutes (e.g., ions) in the liquid, the polymer (e.g.,chemical structure of the monomers), or labels on the polymer. Exemplarytransport properties include current, conductance, resistance,capacitance, charge, concentration, optical properties (e.g.,fluorescence and Raman scattering), and chemical structure.

Desirably, the transport property is current. Suitable methods fordetecting current in aperture containing systems are known in the art,for example, as described in U.S. Pat. Nos. 6,746,594, 6,673,615,6,627,067, 6,464,842, 6,362,002, 6,267,872, 6,015,714, and 5,795,782 andU.S. Publication Nos. 2004/0121525, 2003/0104428, and 2003/0104428, eachof which is incorporated by reference in its entirety. In anotherembodiment, the transport property is electron flow across the aperture,which may be monitored by electrodes disposed adjacent to or abuttingthe aperture boundary.

Additional methods of detecting the interaction of compounds with poreproteins suitable for use in the devices and methods of the inventionare outlined in Bayley and Cremer, Nature 413:226-229 (2001) which ishereby incorporated by reference in its entirety.

Media

The medium disposed in the compartments on either side of the graphenemay be any fluid that permits adequate substrate interaction (e.g.,polynucleotide mobility). Typically, the medium is a liquid, usuallyaqueous solutions or other liquids or solutions in which the polymers orcompounds can be distributed. When an electrically conductive medium isused, it can be any medium that is able to carry electrical current.Such solutions generally contain ions as the current-conducting agents(e.g., sodium, potassium, chloride, calcium, magnesium, cesium, barium,sulfate, or phosphate). Conductance across or through the aperture canbe determined by measuring the flow of current across or through theaperture via the conducting medium. Alternatively, an electrochemicalgradient may be established by a difference in the ionic composition ofthe two pools of medium, either with different ions in each pool, ordifferent concentrations of at least one of the ions in the solutions ormedia of the pools. Conductance changes are measured by the detectionsystem.

Methods of Characterizing Polymers

In general, the invention features characterization of polymersinvolving the use of two separate pools of a medium and an interfacebetween the pools. The aperture between the pools is typically capableof interacting sequentially with the individual monomer residues of apolymer present in one of the pools. Measurements of transportproperties are continued over time, as individual monomer residues ofthe polymer interact sequentially with the aperture, yielding datasuitable to determine a monomer-dependent characteristic of the polymer.The monomer-dependent characterization achieved by sequencing mayinclude identifying characteristics such as, but not limited to, thenumber and composition of monomers that make up each individual polymer,in sequential order.

The polymer being characterized may remain in its original pool or it,or a reaction product including it or fragments thereof, may cross thepore into the other pool. In either situation, the polymer moves inrelation to the pore, and individual monomers interact sequentially withthe pore, giving rise to a change in the measured transport properties,e.g., conductance, of the pore. When the polymer does not cross into thetrans side of the device, it is held adjacent the pore such that itsmonomers interact with the pore passage and bring about the changes intransport properties, which are indicative of polymer characteristics.

Polymers can be driven through the pore with or without the assistanceof a molecular motor. The rate is controlled by the use of a molecularmotor that moves a polymer at a substantially constant rate, at leastfor a portion of time during a characterization. In the absence of amolecule motor, the polymer can be driven through the pore using, e.g.,an electric current or pressure.

Methods of Identifying Transmembrane Protein-Interacting Compounds

The bilayers of the invention can also be used to identify compoundsthat interact with membrane molecules (e.g., pore-forming proteins ortransmembrane receptors). Here, a property (e.g., electricalconductance) of a transmembrane protein disposed inside a grapheneaperture can be measured in the presence and absence of a candidatecompound. If the addition of a compound changes a property of thetransmembrane protein, the compound is identified as a transmembraneprotein-interacting compound. This method can include contacting thetransmembrane protein with a positive or negative control compound(e.g., a known ligand for a transmembrane receptor). In theseembodiments, a property of the transmembrane protein in the presence ofthe control compound can be measured, and a candidate compound thatenhances or disrupts such a property can be identified as being atransmembrane protein-interacting compound.

In another embodiment, the transmembrane protein can be a pore-formingprotein and candidate compounds can be tested to disrupt the flow ofmolecules through the pore. The flow of molecules through the pore canbe measured using, e.g., a measurement of electric current through oracross the aperture.

Known interacting compounds may also be employed to measure the propertyof transmembrane proteins.

In addition, the methods of the invention may be employed to study theinteraction of compounds with lipids, e.g., glycolipids, lipidsconjugated to other compounds, or other non-protein compounds in thebilayer. The methods of the invention may also be employed to study theability of compounds to pass through a lipid bilayer or monolayer, e.g.,in transfection or drug delivery, either directly through the lipidlayer or through a pore in the layer. Such compounds could be free insolution or part of a complex, e.g., encapsulated in a micelle, vesicle,or liposome.

Example

Liposomes can be triggered to form a lipid bilayer membrane that forms ahigh resistance giga-ohm seal across an aperture in a graphene membranethat separates two solution filled chambers (FIG. 5).

A 0.5×0.5 mm CVD grown sheet of graphene was mounted over a 200×200 nmaperture in a 250 nm thick, free-standing, insulating SiN_(x) layer on asilicon substrate frame as shown in FIG. 2. The chip-mounted membranewas inserted in a fluidic cell such that it separated two compartments,each subsequently filled with ionic solutions (250 mM KCl, 2.5 mM TRIS,pH 7.8) in electrical contact with Ag/AgCl electrodes. The resistance ofthe aperture to ionic current flow was then measured by sweeping thevoltage bias between the two chambers separated by the chip-mountedgraphene from −100 mV to +100 mV. Before adding liposomes, the apertureresistance was 46 MΩ, which is the expected access resistance of an 8 nmaperture in graphene. Adding liposomes to the chamber on one side of thegraphene (not shown) or to the chambers on both sides of the graphenehad little effect until lipid fusion was triggered by addition of 2 mMCa⁺⁺. An increased resistance to 720 MΩ was observed within secondsafter addition of Ca⁻⁺, although incubation for four hours at roomtemperature further increased the resistance to over 3.6 GΩ (FIG. 6).

Other Embodiments

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each independent publication or patent application was specificallyand individually indicated to be incorporated by reference.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure that come within known or customary practice withinthe art to which the invention pertains and may be applied to theessential features hereinbefore set forth, and follows in the scope ofthe appended claims.

Other embodiments are in the claims.

What is claimed is:
 1. A device comprising an artificial membrane ongraphene with one or more apertures, wherein said artificial membranecomprises two lipid monolayers, each contacting one face of saidgraphene, and wherein the two lipid monolayers form a lipid bilayerwithin said one or more apertures in said graphene.
 2. The device ofclaim 1, wherein said lipid bilayer further comprises a protein.
 3. Thedevice of claim 2, wherein said protein is a transmembrane protein. 4.The device of claim 3, wherein said transmembrane protein is selectedfrom the group consisting of ion channels, multidrug resistance pumps,cytokine receptors, receptors from the immunoglobin superfamily,receptors from the tumor necrosis factor receptor family, chemokinereceptors, receptor tyrosine kinases, and TGF beta receptors.
 5. Thedevice of claim 1, further comprising at least one pore disposed in atleast one of said one or more apertures, wherein at most one pore isdisposed in each of said one or more apertures.
 6. The device of claim5, further comprising a molecular motor, wherein said motor is adjacentto at least one of said one or more pores and is capable of moving apolymer with respect to the at least one of said one or more pores. 7.The device of claim 6, wherein the molecular motor comprises a DNApolymerase, a RNA polymerase, a ribosome, an exonuclease, or a helicaseand said polymer is a polynucleotide.
 8. The device of claim 7, whereinthe DNA polymerase is selected from E. coli DNA polymerase I, E. coliDNA polymerase I Large Fragment (Klenow fragment), phage T7 DNApolymerase, Phi-29 DNA polymerase, Thermus aquaticus (Taq) DNApolymerase, Thermus flavus (Tfl) DNA polymerase, Thermus Thermophilus(Tth) DNA polymerase, Thermococcus litoralis (Tli) DNA polymerase,Pyrococcus furiosus (Pfu) DNA polymerase, Bacillus stearothermophilus(Bst) DNA polymerase, AMV reverse transcriptase, MMLV reversetranscriptase, and HIV-1 reverse transcriptase.
 9. The device of claim7, wherein the RNA polymerase is selected from T7 RNA polymerase, T3 RNApolymerase, SP6 RNA polymerase, and E. coli RNA polymerase.
 10. Thedevice of claim 7, wherein the exonuclease is selected from exonucleaseLambda, T7 Exonuclease, Exo III, RecJ₁ Exonuclease, Exo I, and Exo T.11. The device of claim 7, wherein the helicase is selected from E-colibacteriophage T7 gp4 and T4 gp41 gene proteins, E. coli protein DnaB, E.coli protein RuvB, and E. coli protein rho.
 12. The device of claim 5,wherein at least one of said pores is α-haemolysin.
 13. The device ofclaim 1, further comprising a frame supporting said graphene.
 14. Thedevice of claim 13, further comprising first and second electrodesdisposed on opposite sides of said graphene.
 15. The device of claim 13,wherein said frame comprises a ceramic or silicon nitride.
 16. Thedevice of claim 1, wherein said device comprises a plurality ofapertures arranged in an array.
 17. The device of claim 16, furthercomprising a frame positioned on one face of the graphene, wherein saidframe forms separate compartments for housing liquid for each of saidapertures.
 18. The device of claim 17, wherein said frame isnon-conductive.
 19. The device of claim 17, wherein said frame comprisesa ceramic or silicon nitride.
 20. The device of claim 17, wherein eachof said compartments further comprises an electrode, and wherein saiddevice further comprises at least one electrode on the side of saidgraphene opposite said compartment.
 21. A method of analyzing a targetpolymer comprising introducing the target polymer to the device of claim5, allowing the target polymer to move with respect to the pore toproduce a signal, and monitoring the signal corresponding to themovement of the target polymer with respect to the pore, therebyanalyzing the target polymer.
 22. The method of claim 21, wherein thesignal monitoring comprises measuring a monomer-dependent characteristicof the target polymer while the target polymer moves with respect to thepore.
 23. The method of claim 22, wherein the monomer dependent propertyis the identity of a monomer or the number of monomers in the polymer.24. The method of claim 20, further comprising altering the rate ofmovement of the polymer before, during, or after the signal monitoring.25. A method of identifying a membrane molecule-interacting compoundcomprising contacting the membrane molecule of the device of claim 1with a compound and measuring at least one activity of said membranemolecule in the presence of said compound, whereby a change in activityin the presence of said compound indicates that said compound interactswith said membrane molecule.
 26. The method of claim 25, wherein anelectric field is applied and said measuring at least one activity ofsaid membrane molecule comprises measuring the electrical resistance ofsaid membrane molecule.
 27. The method of claim 25, wherein saidmembrane molecule is a transmembrane protein.